Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Title: Therapeutic Immune Modulation Against Solid Cancers with Intratumoral Poly-ICLC: A Pilot Trial Running Title: Intratumoral Poly-ICLC in Solid Cancer Treatment Authors: Chrisann Kyi1*, Vladimir Roudko1*, Rachel Sabado1, Yvonne Saenger2, William Loging1, John Mandeli1, Tin Htwe Thin1, Deborah Lehrer1, Michael Donovan1, Marshall Posner1, Krzysztof Misiukiewicz1, Benjamin Greenbaum1, Andres Salazar3, Philip Friedlander1, Nina Bhardwaj1 Tisch Cancer Center, Icahn School of Medicine at Mount Sinai, New York, NY1; Columbia University Medical Center, New York, NY2; Oncovir, Inc, Washington, DC 3 Grant Support: This research was supported by grants from the Cancer Research Institute, the Melanoma Research Alliance and NIH. N.B. is a member of the Parker Institute for Cancer Immunotherapy, which supported the Mount Sinai Hospital Cancer Immunotherapy Program. Disclosures of Potential Conflicts of Interest: N B. is on the scientific advisory board of Neon, CPS companion diagnostics, Genentech and Curevac, Inc. R.S. works as clinical scientist at Genentech, Inc. A.S. is the scientific director of Oncovir, Inc. No potential conflicts of interest were disclosed by other authors. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 TRANSLATIONAL RELEVANCE We present a novel vaccine approach using intratumoral poly-ICLC, a viral mimic of the double-stranded RNA in viral replication, as a strategy of “autovaccination,” i.e. the use of the tumor itself as the antigen source in-situ. In this first evaluation of poly-ICLC in melanoma and head and neck cancer patients, we investigate the safety and tolerability of poly-ICLC administered intratumorally to induce tumor immune infiltration and intramuscularly to induce systemic inflammation. Treatment was well-tolerated with minimal toxicities noted. In the one patient with clinical benefit (stable disease), there was evidence of upregulation of genes associated with chemokine activity, T cell activation and antigen presentation (RNA sequencing), and increased CD4, CD8, PD1 and PDL1 levels (quantitative immunohistochemistry) compared to patients with progressive disease. Although only one patient demonstrated clinical benefit, these findings prompt further investigation into optimal dosing and delivery of intratumoral poly-ICLC, and combinations with immune checkpoint blockade and/or other immunomodulators. ABSTRACT Purpose: Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC), a synthetic double-stranded RNA complex, is a ligand for toll-like receptor-3 (TLR3) and MDA-5 that can activate immune cells such as dendritic cells and trigger NK cells to kill tumor cells. Methods: In this pilot study, eligible patients included those with recurrent metastatic disease who failed prior systemic therapy (head and neck squamous cell cancer (HNSCC), melanoma). Patients received 2 treatment cycles, each cycle consisting of 1mg Poly-ICLC 3x weekly intratumorally (IT) for 2 weeks followed by intramuscular (IM) boosters biweekly for 7 weeks with a 1-week rest period. Immune response was evaluated by immunohistochemistry (IHC) and RNA Sequencing (RNASeq) in tumor and blood. Results: Two patients completed 2 cycles of IT treatments and one achieved clinical benefit (stable disease, PFS 6 months), while the remainder had progressive disease. Poly-ICLC was well tolerated with principal side effects of fatigue and inflammation at injection site (< grade 2). In the patient with clinical benefit, IHC analysis of tumor showed increased CD4, CD8, PD1 and PDL1 levels compared to patients with progressive disease. RNASeq analysis of the same patient’s tumor and PBMC showed dramatic changes in response to Poly-ICLC treatment including upregulation of genes associated with chemokine activity, T cell activation and antigen presentation. Conclusions: Poly-ICLC was well tolerated in solid cancer patients, and generated local and systemic immune responses as evident in the patient achieving clinical benefit. These results warrant further investigation, and are currently being explored in a multicenter phase II clinical trial (NCT02423863). Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 INTRODUCTION The last decade has ushered in an exciting new age of immunotherapy with the FDA approvals of the first cancer vaccine, Provenge or sipuleucel-T, and checkpoint blockade, e.g. ipilimumab (Yervoy; anti- CTLA-4), pembrolizumab (Keytruda; anti-programmed death (PD1)), and nivolumab (Opdivo; anti-PD1). However, even with advances in immune checkpoint blockade and other systemic chemotherapies, there remain a significant fraction of patients who either fail to respond or become resistant to treatment. Proposed interventions to broaden the fraction of patients benefiting from immunotherapies and increase response rates rely on reversing T cell exhaustion, reducing immune suppression in the tumor microenvironment (TME), and transforming a non-inflamed TME to a “responsive” TME (e.g. immune cell infiltration, upregulation of PDL-1 etc). Oncolytic viruses in the treatment of cancer work presumably through their effects not only upon tumor cells but by activating innate immunity and inducing tumor specific immunity 1-4. An intratumoral approach that mimics viral infection, without associated significant side effects or the complications of inducing dominant antiviral immunity, is one proposed strategy5. Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (Poly-ICLC, Hiltonol, Oncovir, Inc) is a synthetic double-stranded RNA viral mimic for a pathogen associated molecular pattern (PAMP) or ‘danger signal’ that binds to toll-like receptor 3 (TLR3), MDA5 and other pathogen receptors to activate dendritic cells (DCs) and subsequently to also trigger NK cells to kill tumor cells. While initially developed as an IFN inducer, Poly-ICLC has been found to have much broader biological effects, including specific anti-tumor and anti-viral actions6. It activates multiple elements of innate and adaptive immunity, including induction of a ‘natural mix’ of IFNs, other cytokines and chemokines, NK cells, T cells, myeloid DCs, the P68 protein kinase (PKR), and other dsRNA-dependent host defense systems7,8. Thus, when properly combined with antigen, Poly-ICLC has the potential to generate a 'live virus vaccine equivalent' with a comprehensive immune response that includes activation of myeloid DCs, other antigen-presenting cells, and NK cells, and generation of a polyfunctional Th1-polarized and CTL response with increase in CD8 to CD4 / regulatory T cell ratio, which via the induction of specific chemokines can home to tumor or pathogen 9-14. While most cancer vaccines are generally designed to utilize known or presumptive tumor antigens, an alternative strategy is ‘autovaccination,’ i.e. the use of the tumor itself as the antigen source, in-situ. Poly- ICLC can be given intramuscularly (IM) to induce systemic inflammation and/or intratumorally (IT) to induce immune infiltration of tumors. We observed a dramatic response in the first sarcoma case being treated with repeated IT and IM Poly-ICLC, an 18-year-old patient with a malignant embryonal rhabdomyosarcoma, who failed eight different regimens of chemotherapy as well as radiation and proton- beam therapy, and was in hospice. Treatment with Poly-ICLC resulted in necrosis and regression of facial, oral, retro-orbital and a large intracerebral tumor. Although the patient eventually succumbed, his life was extended well beyond expectations 15. A phase II trial of single-dose treatment of ultrasound guided IT Poly-ICLC followed by IM Poly-ICLC was found to be safe in patients with advanced primary or metastatic liver cancers, with evidence of regression of non-injected metastatic lesions as well as the targeted lesions16,17. Based upon these early indications of clinical response, we conducted a pilot study using this autovaccination strategy with intratumoral and intramuscular Poly-ICLC at our institution in advanced treatment refractory head and neck cancers and melanoma. We hypothesize that the therapeutic in-situ autovaccination strategy using intratumoral (IT) and intramuscular (IM) Poly-ICLC administration of TLR3 ligand Poly-ICLC can reverse DC inhibition in the treated tumor micro-environment, increase the efficiency of antigen presentation to CTLs, prevent tolerization to tumor antigens, and elicit systemic anti- tumor immunity. Here, we present the first-ever published results of our phase I trial of intratumoral poly- ICLC in treatment of solid cancer patients. Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 27, 2018; DOI: 10.1158/1078-0432.CCR-17-1866 Author manuscripts have been peer reviewed and accepted for publication